RSV vaccines and methods of production and use thereof

ABSTRACT

Recombinant, live, attenuated viruses of the Pneumoviridae family are disclosed that include a baculovirus GP64 envelope glycoprotein or variant or fragment thereof and a respiratory syncytial virus (RSV) F protein variant or fragment thereof. Also disclosed are polynucleotides encoding the virus as well as pharmaceutical compositions and vaccines containing the virus. In addition, methods of producing and using each of the above compositions are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS/INCORPORATION BY REFERENCESTATEMENT

This application claims benefit under 35 USC § 119(e) of provisionalapplication U.S. Ser. No. 62/621,685, filed Jan. 25, 2018. The entirecontents of the above-referenced application are expressly incorporatedherein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with U.S. Government support under NIH Grant No.1R21AI128520-01A1 awarded by the Department of Health and HumanServices. The Government has certain rights in this invention.

BACKGROUND

Respiratory Syncytial Virus (RSV) is the single largest viral cause ofpediatric bronchiolitis and pneumonia, with a high worldwide mortality.In spite of many years of clinical trials and scientific progress, asafe and effective vaccine against RSV has still not been found. In the1960s, a formalin-inactivated RSV vaccine (FI-RSV) induced an imbalancein the immune response which led to enhanced pathology after exposure towild type RSV (known as vaccine-enhanced disease (VED)). Ever since thisencounter with VED, it has been enormously challenging to impart bothsufficient safety and efficacy in a single vaccine. Furthermore, thereare age-specific challenges, and it is generally believed that differentvaccine platforms will be needed for different populations and/or agegroups to lessen the RSV-associated disease burden. For RSV-naïvechildren, live-attenuated vaccines are an important focus, becauseinactivated and subunit vaccines are poor at inducing cell-mediatedimmunity, and this is known to contribute to VED. Moreover, livevaccines typically can also induce broad systemic and local immunity.Thus, for RSV-naïve individuals, a live vaccine approach is anattractive option, provided the vaccine itself is sufficiently safe andcannot revert to a more aggressive phenotype.

RSV contains a negative-sense, single-stranded RNA genome that expresseseleven known proteins from ten genes (FIG. 2). Of these, the attachment(G) and fusion (F) proteins have been characterized as transmembrane(surface) glycoproteins and contain the major antigenic epitopes ofhuman respiratory syncytial virus; as such, the G and F proteins appearto be critical for induction of neutralizing anti-RSV antibodies. Incontrast to G, F is essential for virus infectivity.

U.S. Pat. No. 7,588,770 to Oomens et al., the entire contents of whichare hereby expressly incorporated herein by reference, describesgenetically modified RSVs generated by replacing genes encoding proteinssuch as F and G with genes encoding heterologous envelope proteins,e.g., a baculovirus GP64 envelope glycoprotein. Such geneticallymodified RSVs exhibit improved temperature stability and in some casesare infectious but incapable of cell-to-cell transmission. Thus, theseattenuated viruses are safe for use in vaccines. However, a disadvantageof this technology is that removal of the F and G proteins from thevirus greatly reduces antigenicity, thereby decreasing the ability ofthe viruses to elicit a robust, protective immune response.

It has recently been recognized that the viral fusion (F) protein isunstable and readily shifts to the post-fusion conformation duringpurification or vaccine preparation. As a result, a large proportion ofvaccine-induced antibodies (Abs) target the post-fusion form, which isfunctionally obsolete. To avoid induction of anti-post-fusion F Abs,McLellan et al. were able to genetically stabilize the pre-fusion form(referred to as PreF), thereby greatly increasing neutralizing capacityof anti-F Abs when given as a protein vaccine (see, for example, USPatent Application Publication Nos. US 2015/0030622 (published Jan. 29,2015 to Marshall et al.); US 2016/0031972 (published Feb. 4, 2016 toZheng et al.); and US 2016/0046675 (published Feb. 18, 2016 to Kwong etal.); the entire contents of each of which are hereby expresslyincorporated herein by reference). However, subunit vaccines are deemedunsafe for the RSV-naïve target population. In addition, stabilizationof PreF renders it non-functional, and a virus solely expressing PreF isnot viable.

Therefore, there is a need in the art for new and improved RSV vaccinesthat overcome the disadvantages and defects of the prior art. It is tosuch new and improved vaccines, as well as methods of production and usethereof, that the present disclosure is directed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates three versions of pre-fusion stabilized F proteinvariants that were generated for use in accordance with the presentdisclosure. These pre-fusion stabilized F protein variants are based onthe previously described preF fusion protein variant DS-Cav-1 (see, forexample, US 2015/0030622, US 2016/0031972, and US 2016/0046675,incorporated supra; and McLellan et al. (Science (2013) 342:592-598);the entire contents of which are expressly incorporated herein byreference). preF^(ΔCT) is a membrane-anchored version that is expressedand anchored at the surface of infected cells. RSV-preF^(SEC) is asecreted version that is secreted to the extracellular environment oninfected cells. RSV-preF^(SEC/tag) is similar to RSV-preF^(SEC) butcontains an epitope tag for easy identification and detection. TheDS-Cav-1 mutations are shown by vertical lines. A wildtype F ORF isshown for comparison (574 amino acids). TMD=transmembrane domain.Tag=epitope tag.

FIG. 2 graphically depicts the engineering of RSV viruses withpre-fusion stabilized F variants at the 8th or 6th genome position.Panel A, the wildtype RSV genome; Panel B, RSV genomes with variants ofpre-fusion stabilized F at the 8^(th) genome position; and Panel C, RSVgenomes with variants of pre-fusion stabilized F at the 6^(th) genomeposition. The 6th genome position is more highly expressed than the 8thgenome position, to enhance the level of pre-fusion F. All viruses havea GFP marker gene for tracking and assay purposes. GFP is not requiredand can be removed if necessary. All viruses also lack expression of thesecreted G protein (indicated as Gmem), which is a known virulencefactor.

FIG. 3 graphically depicts that removal of the cytoplasmic tail (CT)strongly improved cell-surface expression of pre-fusion F. HEp2 cellswere transfected with the indicated F expressing plasmids. The F openreading frames were codon-optimized, as native F sequences expresspoorly in transfected cells. To each well, a plasmid expressing NGFR-mycwas added as a transfection control (NGFR-myc is expressed at the cellsurface). At 46 hour post-transfection, transfected cells were incubatedwith various F antibodies or myc antibody as a control, and relative Fsurface levels were detected using standard ELISA.

FIG. 4 graphically depicts verification of the engineered virusesgenerated as in FIG. 2 as successfully expressing pre-fusion stabilizedF protein variants. Cells infected by virus RSV8-preF^(ΔCT) wereincubated with anti-F and anti-G antibodies at 26 hours post-infection,or mock-infected as a negative control, and subjected to ELISA. Threeantibodies (provided by J S McLellan) were used to detect F. The first,Motavizumab (mota), detects both the pre-fusion and post-fusionconformation of F; the second and third antibodies (D25 and 14402) areknown to detect a different epitope specific only for the pre-fusionconformation of F (site Ø and site V). The G protein was detected atsimilar levels. Abundant levels of pre-fusion F were expressed at thesurface of vaccine-virus infected cells. All F Abs were applied at 0.1μg/ml. Error bars are standard deviation of the mean from triplicatesamples. Viruses RSV8-preF^(SEC) and RSV8-preF^(SEC/tag) have beensimilarly examined and also express pre-fusion F.

FIG. 5 graphically depicts that vaccine candidate RSV6-preF^(ΔCT)induced high surface levels of prefusion-F and G. HEp2 cells wereinfected with the indicated viruses. At 26 hours post-infection,infected cells were incubated with F, G, or N antibodies, and relative Fand G surface levels were determined using ELISA (the N protein is anindicator of viral genomic replication and is shown for normalizationpurposes; to detect N, cells are detergent-permeabilized). Four Fantibodies (provided by J S McLellan) were used to detect F. D25, 14402,and AM14 are specific for prefusion F. AM14 only recognizes correctlytrimerized mature prefusion F. 15576 is specific for the postfusionconformation. Absence of 15576 signal shows that preF^(ΔCT) is entirelyin the prefusion conformation. Abundant levels of conformationallycorrect pre-fusion F were expressed at the surface of vaccine-virusinfected cells. As expected, the G protein was detected at similarlevels. All F Abs were applied at 0.1 μg/ml. Error bars are standarddeviation of the mean from triplicate samples.

FIG. 6 graphically depicts that vaccine candidate RSV6-preF^(SEC/tag)secreted high levels of prefusion F (this is the codon-optimized preFgene). HEp2 cells were infected with the indicated viruses. At 36 hourspost-infection, supernatants of infected cells were harvested andincubated on ELISA plates coated with the anti-tag antibodies for 1hour. Bound preF was then detected by ELISA using D25 and motavizumab asprimary antibodies.

FIG. 7 graphically depicts that preF expressing single cycle RSV inducedhigh levels of anti-RSV antibodies in vivo. 96 well plates were coatedwith preF+G by infecting HEp-2 cells with RSV6-preF^(ΔCT). At 26 hourspost-infection, preF and G proteins were present at the cell surface inconformationally accurate (native) form (as shown in FIG. 5). Pooledsera (n=3, collected at 3 weeks post-boost) from mice vaccinatedprime/boost with RSV6-preF^(ΔCT) or RecWT virus were incubated on thecoated ELISA plates, and antibody levels were determined using ELISA.

FIG. 8 graphically depicts a schematic overview of examples of differentpreF-based single cycle RSV vaccines. First panel, RSV6-preF^(ΔCT);second panel, RSV6-preF^(ΔCT)-NS1low; third panel,RSV6-preF^(ΔCT)-NS2low; fourth panel, RSV1-preF^(ΔCT)-NS1low; fifthpanel, RSV2-preF^(ΔCT)-NS2low.

FIG. 9 graphically depicts that different preF RSV vaccines induced highlevels of preF and G protein at the cell surface. HEp-2 cells wereinfected with viruses RSV6-preF^(ΔCT) and RSV1-preF^(ΔCT)-NS1low. At 26hours post-infection, infected cells were incubated with anti-preF andanti-G antibodies, which were subsequently detected by standard ELISAmethod. Anti-N antibody was also used as an indicator for viral genomicreplication (N encapsulates the viral genome), and preF and G antibodylevels were determined without and with N level-based normalization.

FIG. 10 graphically depicts that different preF RSV vaccines inducedhigh levels of preF-specific and G-specific antibodies in vivo. 96 wellplates were coated with preF+G or preF alone as follows: HEP-2 cellswere infected with RSV6-preF^(ΔCT) or RSV6-preF^(ΔCT)-AG (G generemoved). At 26 hours post-infection, either preF+G or preF alone werepresent at the cell surface in conformationally accurate (native) form(as shown in FIG. 5). Pooled sera (n=3, collected at 3 weeks post-boost)from mice vaccinated prime/boost with RSV6-preF^(ΔCT) andRSV1-preF^(ΔCT)-NS1low were incubated on the coated ELISA plates (preF+Gon the left; preF alone on the right), and antibody levels weredetermined using standard ELISA method. Anti-G Ab L9 was used to verifythe absence of G protein in the preF-alone ELISA.

FIG. 11 graphically depicts that two distinct preF RSV vaccines inducedhigher neutralizing antibody activity than a wildtype virus, despitebeing designed as safe, single-cycle vaccines. Three-fold dilutions ofpooled mice sera (3 mice per pool; sera harvested 3 weeks post-boost)were incubated with 500 PFU of virus RSV-AG-HRP, which lacks the Gprotein (allowing detection of F-specific neutralization) and containsthe HRP gene for detection. After a one hour incubation, virus-antibodysuspensions were incubated on HEp-2 cells for 1.5 hours. Inoculum wasremoved and cells incubated for a total of 48 hours post-infection(hpi). At 48 hpi, medium was replaced with standard (TMB) ELISAsubstrate, and OD₄₅₀ was determined after 30 minutes as a measure ofvirus replication.

FIG. 12 graphically depicts that the preF RSV vaccine RSV6-preF^(ΔCT)(codon-optimized PreF) induced higher neutralizing antibody activitythan a wildtype virus, despite being designed as a safe, single-cyclevaccine. Three-fold dilutions of pooled mice sera (3 mice per pool; seraharvested 3 weeks post-boost) were incubated with 250 PFU of virusRSV-AG-HRP, which lacks the G protein (allowing detection of F-specificneutralization) and contains the HRP gene for detection. After a onehour incubation, virus-antibody suspensions were incubated on HEp-2cells for 1.5 hours. Inoculum was removed and cells incubated for atotal of 48 hours post-infection (hpi). At 48 hpi, medium was replacedwith standard (TMB) ELISA substrate, and OD₄₅₀ was determined after 30minutes as a measure of virus replication.

DETAILED DESCRIPTION

Before explaining at least one embodiment of the inventive concept(s) indetail by way of exemplary language and results, it is to be understoodthat the inventive concept(s) is not limited in its application to thedetails of construction and the arrangement of the components set forthin the following description. The inventive concept(s) is capable ofother embodiments or of being practiced or carried out in various ways.As such, the language used herein is intended to be given the broadestpossible scope and meaning; and the embodiments are meant to beexemplary—not exhaustive. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

Unless otherwise defined herein, scientific and technical terms used inconnection with the presently disclosed inventive concept(s) shall havethe meanings that are commonly understood by those of ordinary skill inthe art. Further, unless otherwise required by context, singular termsshall include pluralities and plural terms shall include the singular.The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor chemical syntheses and chemical analyses.

All patents, published patent applications, and non-patent publicationsmentioned in the specification are indicative of the level of skill ofthose skilled in the art to which this presently disclosed inventiveconcept(s) pertains. All patents, published patent applications, andnon-patent publications referenced in any portion of this applicationare herein expressly incorporated by reference in their entirety to thesame extent as if each individual patent or publication was specificallyand individually indicated to be incorporated by reference.

All of the compositions and/or methods disclosed herein can be made andexecuted without undue experimentation in light of the presentdisclosure. While the compositions and methods of the inventiveconcept(s) have been described in terms of particular embodiments, itwill be apparent to those of skill in the art that variations may beapplied to the compositions and/or methods and in the steps or in thesequence of steps of the methods described herein without departing fromthe concept, spirit, and scope of the inventive concept(s). All suchsimilar substitutions and modifications apparent to those skilled in theart are deemed to be within the spirit, scope, and concept of theinventive concept(s) as defined by the appended claims.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

The use of the term “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” As such, the terms “a,” “an,” and “the”include plural referents unless the context clearly indicates otherwise.Thus, for example, reference to “a compound” may refer to one or morecompounds, two or more compounds, three or more compounds, four or morecompounds, or greater numbers of compounds. The term “plurality” refersto “two or more.”

The use of the term “at least one” will be understood to include one aswell as any quantity more than one, including but not limited to, 2, 3,4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term “at least one” mayextend up to 100 or 1000 or more, depending on the term to which it isattached; in addition, the quantities of 100/1000 are not to beconsidered limiting, as higher limits may also produce satisfactoryresults. In addition, the use of the term “at least one of X, Y, and Z”will be understood to include X alone, Y alone, and Z alone, as well asany combination of X, Y, and Z. The use of ordinal number terminology(i.e., “first,” “second,” “third,” “fourth,” etc.) is solely for thepurpose of differentiating between two or more items and is not meant toimply any sequence or order or importance to one item over another orany order of addition, for example.

The use of the term “or” in the claims is used to mean an inclusive“and/or” unless explicitly indicated to refer to alternatives only orunless the alternatives are mutually exclusive. For example, a condition“A or B” is satisfied by any of the following: A is true (or present)and B is false (or not present), A is false (or not present) and B istrue (or present), and both A and B are true (or present).

As used herein, any reference to “one embodiment,” “an embodiment,”“some embodiments,” “one example,” “for example,” or “an example” meansthat a particular element, feature, structure, or characteristicdescribed in connection with the embodiment is included in at least oneembodiment. The appearance of the phrase “in some embodiments” or “oneexample” in various places in the specification is not necessarily allreferring to the same embodiment, for example. Further, all referencesto one or more embodiments or examples are to be construed asnon-limiting to the claims.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for acomposition/apparatus/device, the method being employed to determine thevalue, or the variation that exists among the study subjects. Forexample, but not by way of limitation, when the term “about” isutilized, the designated value may vary by plus or minus twenty percent,or fifteen percent, or twelve percent, or eleven percent, or tenpercent, or nine percent, or eight percent, or seven percent, or sixpercent, or five percent, or four percent, or three percent, or twopercent, or one percent from the specified value, as such variations areappropriate to perform the disclosed methods and as understood bypersons having ordinary skill in the art.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”), or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AAB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, the term “substantially” means that the subsequentlydescribed event or circumstance completely occurs or that thesubsequently described event or circumstance occurs to a great extent ordegree. For example, when associated with a particular event orcircumstance, the term “substantially” means that the subsequentlydescribed event or circumstance occurs at least 80% of the time, or atleast 85% of the time, or at least 90% of the time, or at least 95% ofthe time. For example, the term “substantially adjacent” may mean thattwo items are 100% adjacent to one another, or that the two items arewithin close proximity to one another but not 100% adjacent to oneanother, or that a portion of one of the two items is not 100% adjacentto the other item but is within close proximity to the other item.

The term “polypeptide” as used herein will be understood to refer to apolymer of amino acids. The polymer may include d-, l-, or artificialvariants of amino acids. In addition, the term “polypeptide” will beunderstood to include peptides, proteins, and glycoproteins.

The term “polynucleotide” as used herein will be understood to refer toa polymer of two or more nucleotides. Nucleotides, as used herein, willbe understood to include deoxyribose nucleotides and/or ribosenucleotides, as well as artificial variants thereof. The termpolynucleotide also includes single-stranded and double-strandedmolecules.

The terms “analog” or “variant” as used herein will be understood torefer to a variation of the normal or standard form or the wild-typeform of molecules. For polypeptides or polynucleotides, an analog may bea variant (polymorphism), a mutant, and/or a naturally or artificiallychemically modified version of the wild-type polynucleotide (includingcombinations of the above). Such analogs may have higher, full,intermediate, or lower activity than the normal form of the molecule, orno activity at all. Alternatively and/or in addition thereto, for achemical, an analog may be any structure that has the desiredfunctionalities (including alterations or substitutions in the coremoiety), even if comprised of different atoms or isomeric arrangements.

As used herein, the phrases “associated with” and “coupled to” includeboth direct association/binding of two moieties to one another as wellas indirect association/binding of two moieties to one another.Non-limiting examples of associations/couplings include covalent bindingof one moiety to another moiety either by a direct bond or through aspacer group, non-covalent binding of one moiety to another moietyeither directly or by means of specific binding pair members bound tothe moieties, incorporation of one moiety into another moiety such as bydissolving one moiety in another moiety or by synthesis, and coating onemoiety on another moiety, for example.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 percent of allmacromolecular species present in the composition, more preferably morethan about 85%, 90%, 95%, and 99%. Most preferably, the object speciesis purified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

The term “pharmaceutically acceptable” refers to compounds andcompositions which are suitable for administration to humans and/oranimals without undue adverse side effects such as (but not limited to)toxicity, irritation, and/or allergic response commensurate with areasonable benefit/risk ratio.

The term “patient” as used herein includes human and veterinarysubjects. “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including (but not limited to) humans, domesticand farm animals, nonhuman primates, and any other animal that hasmammary tissue.

The term “child” is meant to refer to a human individual who would berecognized by one of skill in the art as an infant, toddler, etc., or anindividual less than about 18 years of age, usually less than about 16years of age, usually less than about 14 years of age, or even less(e.g., from newborn to about 2, about 3, about 4, about 5, about 6,about 7, about 8, about 9, about 10, about 11, or about 12 years ofage). The term “elderly” generally refers to a human individual whoseage is greater than about 50 years of age, usually greater than about 55years of age, frequently greater than about 60 years of age or more(e.g., about 65 years of age and upwards).

The term “treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Those in need of treatmentinclude, but are not limited to, individuals already having a particularcondition/disease/infection as well as individuals who are at risk ofacquiring a particular condition/disease/infection (e.g., those needingprophylactic/preventative measures). The term “treating” refers toadministering an agent to a patient for therapeutic and/orprophylactic/preventative purposes.

A “therapeutic composition” or “pharmaceutical composition” refers to anagent that may be administered in vivo to bring about a therapeuticand/or prophylactic/preventative effect.

Administering a therapeutically effective amount or prophylacticallyeffective amount is intended to provide a therapeutic benefit in thetreatment, prevention, and/or management of a disease, condition, and/orinfection. The specific amount that is therapeutically effective can bereadily determined by the ordinary medical practitioner, and can varydepending on factors known in the art, such as (but not limited to) thetype of condition/disease/infection, the patient's history and age, thestage of the condition/disease/infection, and the co-administration ofother agents.

The term “effective amount” refers to an amount of a biologically activemolecule or conjugate or derivative thereof sufficient to exhibit adetectable therapeutic effect without undue adverse side effects (suchas (but not limited to) toxicity, irritation, and allergic response)commensurate with a reasonable benefit/risk ratio when used in themanner of the inventive concept(s). The therapeutic effect may include,for example but not by way of limitation, preventing, inhibiting, orreducing the occurrence of infection by or growth of microbes and/oropportunistic infections. The effective amount for a subject will dependupon the type of subject, the subject's size and health, the nature andseverity of the condition/disease/infection to be treated, the method ofadministration, the duration of treatment, the nature of concurrenttherapy (if any), the specific formulations employed, and the like.Thus, it is not possible to specify an exact effective amount inadvance. However, the effective amount for a given situation can bedetermined by one of ordinary skill in the art using routineexperimentation based on the information provided herein.

As used herein, the term “concurrent therapy” is used interchangeablywith the terms “combination therapy” and “adjunct therapy,” and will beunderstood to mean that the patient in need of treatment is treated orgiven another drug for the disease/infection in conjunction with thepharmaceutical compositions of the present disclosure. This concurrenttherapy can be sequential therapy, where the patient is treated firstwith one pharmaceutical composition and then the other pharmaceuticalcomposition, or the two pharmaceutical compositions are givensimultaneously.

The terms “administration” and “administering,” as used herein, will beunderstood to include all routes of administration known in the art,including but not limited to, oral, topical, transdermal, parenteral,subcutaneous, intranasal, mucosal, intramuscular, intraperitoneal,intravitreal, and intravenous routes, and including both local andsystemic applications. In addition, the compositions of the presentdisclosure (and/or the methods of administration of same) may bedesigned to provide delayed, controlled, or sustained release usingformulation techniques which are well known in the art.

Turning now to the inventive concept(s), certain non-limitingembodiments of the present disclosure are directed to a recombinant,live, attenuated virus of the Pneumoviridae family. The recombinant,live, attenuated virus includes a baculovirus GP64 envelope glycoproteinor variant or fragment thereof and a polynucleotide encoding arespiratory syncytial virus (RSV) F protein variant or fragment thereof.The baculovirus G64 envelope glycoprotein or fragment thereof is capableof mediating entry of the recombinant virus into a mammalian cell. Therespiratory syncytial virus (RSV) F protein variant or fragment thereofincludes at least one amino acid substitution when compared to a nativeRSV F protein, wherein the at least one amino acid substitutionstabilizes the RSV F protein variant or fragment thereof in a pre-fusionconformation.

In certain non-limiting embodiments, the recombinant, live, attenuatedvirus is isolated from the cell in which it is produced.

In certain non-limiting embodiments, the recombinant, live, attenuatedvirus is further defined as a recombinant respiratory syncytial virus(RSV).

In certain non-limiting embodiments, the recombinant, live, attenuatedvirus is capable of infecting a cell in a mammal but cannot transmitfrom said cell to another cell in the mammal.

In certain non-limiting embodiments, the recombinant, live, attenuatedvirus is further defined as an enveloped recombinant, live, attenuatedvirus.

Also, in certain non-limiting embodiments, the recombinant, live,attenuated virus maintains infective stability when stored at above 0°C. for at least 3.5 days.

Any baculovirus GP64 envelope glycoprotein, variant thereof, or fragmentthereof known in the art or otherwise contemplated herein may beutilized in accordance with the present disclosure, so long as theprotein/variant/fragment is capable of mediating entry of therecombinant virus into a mammalian cell. In certain particular (butnon-limiting) embodiments, the baculovirus GP64 envelope glycoprotein orvariant or fragment thereof comprises an ectodomain of the baculovirusGP64 envelope glycoprotein, a transmembrane domain of the baculovirusGP64 envelope glycoprotein, and/or a heterologous cytoplasmic tail (suchas, but not limited to, a polypeptide from the F protein (such as, butnot limited to, a 12 amino acid polypeptide). Non-limiting examples ofGP64 proteins/variants/fragments that may be utilized in accordance withthe present disclosure are disclosed in US Patent ApplicationPublication No. 2007/0104734, published May 10, 2007 to Oomens et al.and U.S. Pat. No. 7,588,770, issued Sep. 15, 2009 to Oomens et al., aswell as Oomens et al. (Journal of Virology (2004) 78:9064-9072); theentire contents of each of the above references are hereby expresslyincorporated herein by reference. One particular (but non-limiting)example of a GP64 glycoprotein variant disclosed in the above referencesthat may be utilized in accordance with the present disclosure isGP^(64/F), in which the 7-amino acid cytoplasmic tail domain of GP64 wasreplaced by the 12 C-terminal amino acids of the HRSV F protein; theamino acid sequence of GP^(64/F) is represented by SEQ ID NO:15, and thenucleotide sequence encoding same is represented by SEQ ID NO:16.

In addition, the baculovirus GP64 envelope glycoprotein or variant orfragment thereof may not be directly encoded by the virus but rather isobtained from the cell line from which the virus is produced.Alternatively, the GP64 glycoprotein or variant or fragment thereof maybe encoded by the virus.

Also, the recombinant, live, attenuated virus may further encode atleast one other protein normally encoded by the virus' wild type genome,or may further encode at least one variant or fragment thereof. Forexample (but not by way of limitation), the virus may further encode atleast one of RSV NS1 protein or a variant or fragment thereof; NS2protein or a variant or fragment thereof; N protein or a variant orfragment thereof; P protein or a variant or fragment thereof; M proteinor a variant or fragment thereof; SH protein or a variant or fragmentthereof; G protein or a variant or fragment thereof; M-2 protein or avariant or fragment thereof; L protein or a variant or fragment thereof;or any combination thereof. One particular (but non-limiting) variant orfragment of a genomic protein that may be utilized is the secreted Gprotein (known as Gmem); the amino acid sequence of Gmem is representedby SEQ ID NO:13, and the nucleotide sequence encoding same isrepresented by SEQ ID NO:14. Alternatively, the wild type RSV G proteinmay be present in any of the recombinant, live, attenuated viruses ofthe present disclosure; the gene encoding the wild type RSV G protein isrepresented by SEQ ID NO:17, while a codon-optimized sequence encodingthe wild type RSV G protein is represented by SEQ ID NO:18.

In certain embodiments, the virus may be further defined as lackingexpression of at least one virulence factor encoded by the wild typevirus, such as (but not limited to), the NS1 or NS2 protein or Gmem.

Any RSV F protein variant or fragment thereof known in the art orotherwise contemplated herein may be utilized in accordance with thepresent disclosure, so long as the RSV F protein variant or fragmentthereof includes at least one amino acid substitution compared to anative RSV F protein that stabilizes the RSV F protein variant orfragment thereof in a pre-fusion conformation. Any amino acidsubstitution(s) capable of stabilizing the RSV F proteinvariant/fragment in the pre-fusion confirmation may be utilized inaccordance with the present disclosure. Particular (but non-limiting)examples of RSV F protein variants or fragments thereof that can beutilized in accordance with the present disclosure include RSV F proteinvariants or fragments thereof that include at least one, at least two,at least three, or all four of the amino acid substitutions S155C,S190F, V207L, and S290C when compared to the native RSV F proteinsequence, as represented by SEQ ID NO:1. For example (but not by way oflimitation), the RSV F protein variant or fragment thereof can comprisean amino acid sequence represented by at least one of SEQ ID NOS:2-4(see Table 1 and FIG. 1).

In certain non-limiting embodiments, the RSV F protein variant orfragment thereof is absent a portion or all of a cytoplasmic tail and/ora portion or all of a transmembrane domain of the native RSV F protein.Alternatively, the RSV F protein variant or fragment thereof may includea portion or all of the cytoplasmic tail and/or a portion or all of thetransmembrane domain of the native RSV F protein. In one particular (butnon-limiting) embodiment, the transmembrane domain approximatelycorresponds to residues 525-550 of SEQ ID NO:1, while the cytoplasmictail approximately corresponds to residues 554-574 of SEQ ID NO:1.

Alternatively (and/or in addition thereto), the RSV F protein variant orfragment thereof further comprises at least one epitope tag. Onenon-limiting example of an epitope tag that may be utilized inaccordance with the present disclosure is the AcV5 epitope tag. Theamino acid sequence of the AcV5 epitope tag is represented by SEQ IDNO:11, and the nucleotide sequence of this tag is represented by SEQ IDNO:12.

Alternatively (and/or in addition to thereto), the RSV F protein variantor fragment thereof may further include a detectable marker.

As stated herein above, any amino acid substitution(s) capable ofstabilizing the RSV F protein variant/fragment in the pre-fusionconfirmation may be utilized in accordance with the present disclosure.Non-limiting examples of RSV F protein variants or fragments thereof(that contain one or more amino acid substitution(s) capable ofstabilizing the RSV F protein variant/fragment in the pre-fusionconfirmation) are disclosed in US Patent Application Publication Nos. US2015/0030622, US 2016/0031972, and US 2016/0046675 (all incorporatedsupra); McLellan et al. (Science (2013) 342:592-598); Krarup et al.(Nature Communications (2015) 6:8143 (Pages 1-12); and Joyce et al.(Nature Structural and Molecular Biology (2016) 23:811-822); the entirecontents of these references being expressly incorporated herein byreference.

Any polynucleotide encoding any of the RSV F protein variants orfragments thereof may be utilized in accordance with the presentdisclosure. In certain non-limiting embodiments, the polynucleotidesequence corresponds to the wild type RSV F protein sequence (except forthe codons encoding the amino acid substitution(s)). Alternatively, thepolynucleotide sequence may be codon-optimized to increase expressionthereof in a host cell. For example, as shown in Table 1, SEQ ID NOS:8,9, and 10 contain polynucleotide sequences that encode the variants ofSEQ ID NOS:2, 3, and 4, respectively, and are identical to thecorresponding portion of the nucleotide sequence of the wild type Fprotein sequence, with the exception of the codons encoding for theamino acid substitutions. Alternatively, SEQ ID NOS:5, 6, and 7 alsoencode the variants of SEQ ID NOS:2, 3, and 4, respectively, but thesepolynucleotides have been codon-optimized to increase the expressionthereof in a host cell.

TABLE 1 Nucleotide Nucleotide Sequence RSV F Sequence Based on ProteinBased on WT Codon-Optimized Variant* AA Sequence RSV F Sequence RSV FSequence preF^(ΔCT) SEQ ID NO: 2 SEQ ID NO: 8 SEQ ID NO: 5 preF^(SEC)SEQ ID NO: 3 SEQ ID NO: 9 SEQ ID NO: 6 preF^(SEC/tag) SEQ ID NO: 4  SEQID NO: 10 SEQ ID NO: 7 *See FIG. 1 for the structures of each of the RSVF protein variants

In certain non-limiting embodiments, the recombinant, live, attenuatedvirus further comprises the expressed RSV F protein variant or fragmentthereof (as opposed to simply including the polynucleotide sequenceencoding same).

Certain non-limiting embodiments of the present disclosure are alsodirected to an isolated immunogenic composition comprising any of theviruses described or otherwise contemplated herein.

Further non-limiting embodiments of the present disclosure are directedto at least one cell that is capable of producing any of therecombinant, live, attenuated viruses described or otherwisecontemplated herein. The cell(s) includes: (i) at least onepolynucleotide encoding a baculovirus GP64 envelope glycoprotein orvariant or fragment thereof; and (ii) at least one polynucleotideencoding at least a portion of a genome of an infection-attenuated virusof the Pneumoviridae family, wherein the genome comprises a geneencoding an RSV F protein variant or fragment thereof that comprises atleast one amino acid substitution compared to a native RSV F protein,wherein the at least one amino acid substitution stabilizes the RSV Fprotein variant or fragment thereof in a pre-fusion conformation. Incertain non-limiting embodiments, the RSV genome includes otheradditions or modification thereto.

Any cell type capable of producing the recombinant, live, attenuatedviruses and capable of functioning as described or otherwisecontemplated herein falls within the scope of the present disclosure. Incertain non-limiting embodiments, the cell is a mammalian cell. Incertain particular (but non-limiting) embodiments, the cell is a Vero orHEp-2 cell, or any high-producing cell type such as (but not limited to)293 cells. In a particular (but non-limiting) embodiment, the cell is aVbac cell, which is a Vero cell stably transfected with the baculovirusGP64 protein carrying a portion of the cytoplasmic tail of RSV F protein(i.e., GP^(64/F)) (as disclosed in Oomens et al. (2004); US PatentApplication Publication No. 2007/0104734; and U.S. Pat. No. 7,588,770;incorporated supra). In addition, the present disclosure also includesmodified versions of any of the above cell lines. For example (but notby way of limitation), a Vbac cell line can be modified to includeadditional genetic modifications to GP^(64/F), including one or moremodifications to the GP64 portion of the sequence and/or one or moremodifications to the F cytoplasmic tail portion. Alternatively, othercell lines may be modified to express GP64 or a variant thereof (suchas, but not limited to, GP^(64/F), a modified form thereof, or anothermodified form of GP64). These modified cell lines may be produced toimprove on the growth characteristics of the cell line and/or to improveon the cell line's ability to produce virus, thereby enhancingproduction of the compositions of the present disclosure.

In still yet another aspect, mammalian cells or mammals are providedwhich include a recombinant virus as described or otherwise contemplatedherein, or which include polynucleotide(s) that encode all of thevarious components of the recombinant virus, as described or otherwisecontemplated herein.

In a particular (but non-limiting) embodiment, a mammalian cell of thepresent disclosure includes an expression cassette encoding aheterologous envelope protein comprising an ectodomain of a baculovirustransmembrane protein, and one or more expression vectors comprising orencoding the genome of an infection-defective or infection-attenuatedmammalian virus as known in the art or as described or otherwisecontemplated herein. The expression cassette can be stably ortransiently transfected or transduced into the mammalian cell. In oneexample, the expression cassette is integrated into a chromosome of themammalian cell. In another example, the mammalian cell is a Vero cell.

The infection-defective or -attenuated mammalian virus, when assembledin the mammalian cell, incorporates the heterologous envelope proteinwhich affords the virus with improved infectivity and/or stability. Inone example, the mammalian virus is a recombinant RSV, and theheterologous envelope protein comprises an ectodomain of baculovirusenvelope GP64 protein. In another example, the recombinant RSV lacks oneor more functional transmembrane proteins, such as SH, G, or F proteins.The recombinant RSV also includes a pre-fusion F protein variant asdescribed or otherwise contemplated herein.

Furthermore, certain non-limiting embodiments of the present disclosureare directed to mammalian cells, such as (but not limited to) Verocells, that are stably transfected or transduced with at least oneexpression cassette encoding a recombinant viral envelope protein (orvariant or fragment thereof) and encoding a pre-fusion F proteinvariant. The viral envelope protein includes an ectodomain of abaculovirus transmembrane protein (e.g., the GP64 protein). Thepre-fusion F protein variant is as described or otherwise contemplatedherein. These recombinant RSVs are attenuated for cell-to-celltransmission.

Recombinant mammalian or vertebrate viruses other than pneumoviruses canbe similarly prepared using the present disclosure. These viruses mayhave all of the advantageous properties possessed by the recombinantpneumoviruses that are disclosed or otherwise contemplated herein. Forinstance, these viruses can have improved stability of infectivity ascompared to their wild-type counterparts. In addition, these viruses canbe temperature sensitive and infectious but incapable of spreadingbetween host cells. In one embodiment, these viruses includeheterologous envelope proteins that comprise the ectodomain of abaculovirus transmembrane protein, such as (but not limited to) the GP64protein or its functional equivalents, and a pre-fusion F proteinvariant. In another embodiment, these viruses are not recombinantlentiviruses, such as (but not limited to) those described in Kumar, etal. (Human Gene Therapy (2003) 14:67-77) and Ojala, et al. (Biochem.Biophys. Res. Commun. (2001) 284:777-784), the entire contents of eachof which are hereby expressly incorporated herein by reference.

While certain non-limiting embodiments of the present disclosure aredirected to viral production whereby the baculovirus GP64protein/variant/fragment is supplied in trans to RSV from the cell linein which the virus is produced, it should be understood that the scopeof the present disclosure also includes modifying the RSV genome todirectly contain the gene encoding the GP64 protein/variant/fragment. Inthis manner, the RSV genome encodes both: (i) any of the RSV F proteinvariants/fragments described or otherwise contemplated herein; and (ii)any of the GP64 proteins/variants/fragments described or otherwisecontemplated herein. Having GP64 encoded in the viral genome ensuresstrong GP64 expression levels, which improves virus production and virustemperature stability. When the GP64 protein is provided in cis, the RSVgenome will need to be further modified so as to provide a self-limitedsafety component thereto (i.e., by inactivating one or more essentialviral components). One non-limiting example of such a modification is toreplace the M gene with that of the tet-transactivator gene; theresulting vaccine virus is then amplified in the laboratory by growingin M-expressing cells, whereby M is expressed via tet-responsivepromoters. Another non-limiting example of such a modification is toreplace the M gene with any suitable non-RSV gene, whereby the resultingvaccine virus is then amplified in the laboratory by growing inM-expressing cells, whereby M is expressed via inducible or constitutivepromoters.

Further non-limiting embodiments of the present disclosure are directedto a pharmaceutical composition that includes a therapeuticallyeffective amount of any of the recombinant, live, attenuated virusesdescribed in detail herein above or otherwise contemplated herein.Alternatively and/or in addition thereto, the pharmaceutical compositionmay include any of the polynucleotides described or otherwisecontemplated herein. In certain non-limiting embodiments, thepharmaceutical composition is capable of eliciting an immune responseagainst the virus or a component thereof in a mammal. In particular (butnon-limiting) embodiments, the therapeutically effective amount of therecombinant, live, attenuated virus is further defined as an amountsufficient to induce an immune response protective against RSVinfection. Thus, in a particular (but non-limiting) embodiment, thepharmaceutical composition may be an immunogenic composition, such as(but not limited to) a vaccine.

The pharmaceutical compositions or formulations disclosed or otherwisecontemplated herein include one or more attenuated viruses as describedherein, each of which is substantially purified and/or isolated, exceptthat one or more of such viruses may be included in a singlecomposition. In certain non-limiting embodiments, the pharmaceuticalcompositions also include a pharmaceutically acceptable carrier orexcipient. Any carriers or excipients known in the art may be utilizedin accordance with the present disclosure. For example (but not by wayof limitation), a physiological compatible carrier (e.g., saline) thatis compatible with maintaining the infectivity of the virus whenadministered (i.e., the viruses that are initially administered arecapable of infecting one or more host cells), and compatible with thedesired mode of administration, may be utilized as the pharmaceuticallyacceptable carrier in accordance with the present disclosure. Inaddition, the active ingredients may be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredients.Suitable excipients include, for example but not by way of limitation,water, saline, dextrose, glycerol, ethanol, and the like, or anycombination thereof.

The preparation of such compositions for use as immunogeniccompositions, such as (but not limited to) vaccines, is well known tothose of skill in the art. Typically, such compositions are preparedeither as liquid solutions or suspensions; however, solid forms such as(but not limited to) tablets, pills, powders, and the like are alsocontemplated. Solid forms suitable for solution in, or suspension in,liquids prior to administration may also be prepared. The preparationmay also be emulsified. In addition, the pharmaceutical compositionsdisclosed or otherwise contemplated herein may contain minor amounts ofauxiliary substances, such as (but not limited to) wetting oremulsifying agents, pH buffering agents, and the like, as well as anycombination thereof. If it is desired to administer an oral form of thepharmaceutical composition, one or more of various thickeners,flavorings, diluents, emulsifiers, dispersing aids, binders, or thelike, as well as any combination thereof, may be added. Thepharmaceutical compositions of the present disclosure may contain anysuch additional ingredients so as to provide the composition in a formsuitable for administration.

In addition, in certain non-limiting embodiments, the pharmaceuticalcomposition contains at least one adjuvant. Suitable adjuvants are wellknown to those skilled in the art and include, without limitation,aluminum phosphate; at least one saponin complexed to at least onemembrane protein antigen to produce immune stimulating complex(es)(ISCOMs); at least one plutonic polymer with mineral oil; killedmycobacteria in mineral oil; Freund's complete adjuvant; at least onebacterial product, such as (but not limited to) muramyl dipeptide (MDP)and lipopolysaccharide (LPS); monophoryl lipid A; QS 21; andpolyphosphazene, as well as any component or derivative thereof, and aswell as any combination thereof.

The recombinant, live, attenuated virus may be present in thepharmaceutical composition at any percentage of concentration thatallows the virus to function as described or as otherwise contemplatedherein. For example (but not by way of limitation), the virus may bepresent in a sufficient amount to function as an immunogeniccomposition. In certain particular (but non-limiting) embodiments, therecombinant, live, attenuated virus is present in the pharmaceuticalcomposition at a percent concentration of about 0.001%, about 0.005%,about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 2%,about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%,about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, and about 99%. Inaddition, the scope of the presently disclosure also includes thepresence of the virus in the pharmaceutical composition at any percentconcentration that falls within any range formed from the combination oftwo values listed above (for example, a range of from about 1% to about99%, a range of from about 2% to about 80%, a range of from about 3% toabout 60%, a range of from about 10% to about 95%, a range of from about40% to about 75%, etc.).

Likewise, a pharmaceutically acceptable carrier, excipient, and/oradjuvant may be present in the pharmaceutical composition at anypercentage of concentration that allows the carrier/excipient/adjuvantto function as described or as otherwise contemplated herein. In certainparticular (but non-limiting) embodiments, each of the pharmaceuticallyacceptable carrier, excipient, and adjuvant is present in thepharmaceutical composition at a percent concentration of about 0.001%,about 0.005%, about 0.01%, about 0.05%, about 0.1%, about 0.5%, about1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%,about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,and about 99%. In addition, the scope of the presently disclosure alsoincludes the presence of each of the pharmaceutically acceptablecarrier, excipient, and adjuvant in the pharmaceutical composition atany percent concentration that falls within any range formed from thecombination of two values listed above (for example, a range of fromabout 1% to about 99%, a range of from about 2% to about 80%, a range offrom about 3% to about 60%, a range of from about 10% to about 95%, arange of from about 40% to about 75%, etc.).

The pharmaceutical compositions of the present disclosure may beadministered by any of the many suitable means described herein and/orwhich are well known to those of skill in the art, including but notlimited to: by injection, inhalation, oral, intravaginal, intranasal,rectal, or intradermal administration; by ingestion of a food orprobiotic product containing the virus; by topical administration, suchas (but not limited to) as eye drops, sprays, etc.; and the like. In oneinstance, the administration will be carried out by using an implant. Inparticular (but non-limiting) embodiments, the mode of administration isby injection and/or inhalation. One or more than one route ofadministration can be employed either simultaneously or partially orwholly sequentially, i.e., prime boost vaccine regimens are alsocontemplated. Such prime boost vaccine regimens typically involverepeated vaccine administration at preselected intervals, such as (butnot limited to) at 1 month or 6 weeks of age then at 6 months, 1 year,and yearly thereafter, or at longer intervals, e.g., every 5 or 10years, etc. Those of skill in the art are well acquainted with theplanning, implementation, and assessment of such vaccine strategies, andtherefore no further discussion thereof is required.

The pharmaceutical compositions may be administered in conjunction withother treatment modalities. In some embodiments, such modalities mayinclude (but are not limited to) various substances that boost theimmune system, various chemotherapeutic agents, vitamins, anti-allergyagents, anti-inflammatory agents, etc. In other embodiments, otherantigenic agents (e.g., other vaccines or vaccinogens), may beadvantageously administered or co-administered with the pharmaceuticalcompositions disclosed or otherwise contemplated herein. For example, insome cases it may be desirable to combine any of the recombinant viruspharmaceutical compositions disclosed or otherwise contemplated hereinwith other known vaccines which induce protective responses to otheragents, particularly other childhood viruses or other infectious agents.The other vaccines may also be live attenuated virus vaccines, but thisneed not always be the case; such vaccines may be inactivated virusvaccines or vaccines against other etiological agents (e.g., bacteria).When multiple immunogenic compositions/vaccines are to be administeredtogether, the immunogenic compositions/vaccine agents may be combined ina single pharmaceutical composition. Alternatively (and/or in additionthereto), the multiple immunogenic compositions/vaccines may beadministered separately but over a short time interval, e.g., at asingle visit at a doctor's office or clinic, etc.

In addition, the attenuated viruses disclosed or otherwise contemplatedherein may also be further genetically engineered to contain and expressgenes encoding other antigens and/or agents of interest. The otheragents of interest may, for example (but not by way of limitation),include a foreign epitope or other “tag” or “marker” of heterologous orforeign genetic material. Such agents are useful, for example, fordistinguishing between wildtype and vaccine viral strains, such as (butnot limited to) in the laboratory, in nature, in a host, etc. Basically,a genetically engineered recombinant virus disclosed or otherwisecontemplated herein would carry the tag, but the wildtype virus wouldnot. This technique can also be used to distinguish between viralvaccine strains, permitting the introduction of unique genetic tags intodifferent batches or different iterations of recombinant virus strains,to detect pirated formulations, etc. In addition, the geneticallyengineered viruses may contain and express detectable markers (e.g.,labeling or reporter groups such as (but not limited to) variouspeptides and/or proteins), for the purpose of tracing or visualizing thelocation of the viruses, cells infected by the viruses, or proteinstranslated from the viral genome; or for quantitating viruses or cellsinfected by virus, etc. Exemplary detectable markers include, but arenot limited to: various fluorescent entities such as green fluorescentprotein (GFP), blue, cyan, etc. fluorescent protein, and variousderivatives thereof; other fluorescent proteins such as (but not limitedto) dsRed, eqFP611, Dronpa, TagRFPs, KFP, EosFP, Dendra, IrisFP, etc.;other similar molecules known in the art; and any derivatives orcombinations thereof.

Yet further non-limiting embodiments of the present disclosure aredirected to a method of producing any of the recombinant, live,attenuated viruses described or otherwise contemplated herein. In onenon-limiting embodiment of the method, a cell line is provided thatexpresses a baculovirus GP64 envelope glycoprotein or variant orfragment thereof; the cell line is also transfected with at least onepolynucleotide encoding RSV virus, wherein the RSV virus comprises anRSV F protein variant or fragment thereof that comprises at least oneamino acid substitution compared to a native RSV F protein, wherein theat least one amino acid substitution stabilizes the RSV F proteinvariant or fragment thereof in a pre-fusion conformation. In addition,the cell line is cultured under conditions that allow for production ofthe recombinant, live, attenuated virus. In certain particular (butnon-limiting) embodiments, the recombinant, live, attenuated virus isisolated away from the cultured cells. In a particular (butnon-limiting) embodiment, the recombinant, live, attenuated virus issubstantially purified.

In certain particular (but non-limiting) embodiments, the methodincludes: (i) recovering recombinant, live, attenuated virus comprisinga polynucleotide encoding a respiratory syncytial virus (RSV) F proteinvariant or fragment thereof from cDNA using reverse genetics in thepresence of a baculovirus GP64 envelope glycoprotein or variant orfragment thereof, wherein the RSV F protein variant or fragment thereofcomprises at least one amino acid substitution compared to a native RSVF protein, wherein the at least one amino acid substitution stabilizesthe RSV F protein variant or fragment thereof in a pre-fusionconformation; and (ii) amplifying the attenuated virus in a cell lineexpressing the baculovirus GP64 envelope glycoprotein or variant orfragment thereof.

Yet further non-limiting embodiments of the present disclosure aredirected to a use of any of the recombinant, live, attenuated virusesdisclosed or otherwise contemplated herein for the manufacture of amedication for eliciting an immune response in a mammal. In a particular(but non-limiting) embodiment, the medication so produced is a vaccine.

Additional non-limiting embodiments of the present disclosure aredirected to a method of administering any of the pharmaceuticalcompositions disclosed or otherwise contemplated herein to a subject inneed thereof. The amount of attenuated virus that is administered to asubject in need thereof varies according to many factors, e.g., the age,weight, overall health, gender, genetic history, history of allergies,prior infection, or vaccine history, etc. of the subject. Thepharmaceutical compositions can be administered in a manner compatiblewith the dosage formulation and in such amounts as will betherapeutically effective (e.g., immunogenic and/or protective againstinfection with a wild type virus). The quantity to be administereddepends on the subject to be treated, including, for example, thecapacity of the immune system of the individual to synthesizeantibodies, and, if needed, to produce a cell-mediated immune response.Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner and may be monitored on apatient-by-patient basis. However, suitable dosage ranges are readilydeterminable by one skilled in the art and generally range from about10² to about 10⁹ plaque forming units (PFU) or more of virus perpatient, more commonly, from about 10⁴ to about 10⁵ PFU of virus perpatient. The dosage may also depend, without limitation, on the route ofadministration, the patient's state of health and weight, and the natureof the formulation.

Upon inoculation with the pharmaceutical/vaccine compositions disclosedor otherwise contemplated herein, the immune system of the host canrespond to the vaccine by producing antibodies, both secretory andserum, specific for the epitope(s) included in or expressed by therecombinant viruses. As a result of the vaccination, the host can becomepartially or completely immune to infection by the pathogen(s) carryingthe epitope(s) or to wild type counterparts of the attenuated virusesthat were injected. Where the epitope(s) is associated with human RSV(HRSV), the host may become resistant to developing RSV infection, or todeveloping moderate or severe RSV infection, particularly of the lowerrespiratory tract. The immune response may be innate or adaptive, andmay be either cell-mediated or humoral. In a particular (butnon-limiting) embodiment, the response is adaptive and leads toimmunological memory. In a particular (but non-limiting) embodiment, theresponse is protective, i.e., the response prevents or at least lessensthe impact of (e.g., avoids development of serious symptoms of)infection by other viruses with shared antigens and/or epitopes, e.g.,other Pneumoviridae such as (but not limited to) wild typePneumoviridae. Single or multiple administrations of the pharmaceuticalcomposition disclosed or otherwise contemplated herein can be carriedout. In neonates and infants, multiple administrations may be requiredto elicit sufficient levels of immunity. Administration can begin withinthe first month of life and continue at intervals throughout childhood,such as (but not limited to) at two months, six months, one year, andtwo years, as necessary to maintain sufficient levels of protectionagainst the pathogen of interest. Similarly, adults who are particularlysusceptible to repeated or serious infection by the pathogen ofinterest, such as (but not limited to) health care workers, day careworkers, the elderly, individuals with compromised immune function, andindividuals with compromised cardiopulmonary function, may requiremultiple immunizations to establish and/or maintain protective immuneresponses. Levels of induced immunity can be monitored by measuringamounts of neutralizing secretory and serum antibodies, and dosagesadjusted and/or vaccinations repeated as necessary to maintain desiredlevels of protection.

Subjects who may be immunized using the formulations of pharmaceuticalcompositions disclosed or otherwise contemplated herein are usuallymammals and are frequently humans, particularly human infants orchildren. However, this need not always be the case. Veterinary uses ofthe pharmaceutical compositions and methods disclosed or otherwisecontemplated herein are also contemplated, e.g., for companion pets, orfor animals that are of commercial value e.g., as a food source, or forany other animal, etc.

Further non-limiting embodiments of the present disclosure also includemethods of eliciting an immune response to Pneumoviridae viruses in asubject or patient in need thereof. The method includes a step ofadministering any of the pharmaceutical compositions disclosed orotherwise contemplated herein to a subject. The method may include astep of identifying suitable recipients and/or of evaluating ormonitoring the patient's reaction or response to administration of thecomposition. In some embodiments, the composition comprises a live,recombinant attenuated mammalian (e.g., human) RSV (as described hereinabove or otherwise contemplated herein), and the subject is a child, animmunocompromised individual, an elderly patient, and/or any patient atrisk of being exposed to RSV and developing an RSV infection. The methodmay be a method of vaccinating such individuals against developingsevere (or alternatively, moderate) lower respiratory tract disease,e.g., against developing bronchiolitis.

The recombinant live, attenuated viruses disclosed or otherwisecontemplated herein can also be used in diagnostic applications. In onenon-limiting embodiment, a method useful for detecting the presence orabsence of an antibody specifically reactive with an epitope isprovided. The method includes the steps of contacting a sample with therecombinant virus carrying the epitope, and detecting any bindingbetween an antibody component in the sample and the recombinant virus.Examples of binding assays that are suitable for this purpose include(but are not limited to) ELISA (enzyme-linked immunosorbent assay), RIA(radioimmunoassay), FACS (fluorescence-activated cell sorter), and anycombinations thereof.

Yet further non-limiting embodiments of the present disclosure include amethod of generating antibodies specific for RSV in a mammal, whereinthe method includes introducing into the mammal any of the recombinant,live, attenuated viruses disclosed or otherwise contemplated herein (orany of the pharmaceutical compositions containing same, as disclosed orotherwise contemplated herein). Antibodies which specifically recognizeone of the proteins or fragments thereof present in the virus may beused to detect production of the particular protein(s)/fragment(s),either in a laboratory setting (e.g., for research purposes) and/or tomonitor infections established with the attenuated virus in a subject.Antibodies which specifically recognize the attenuated viruses disclosedor otherwise contemplated herein (both mono- and polyclonal) are alsoencompassed by the present disclosure. In some embodiments, antibodyrecognition is selective rather than specific. Antibodies may bepolyclonal or monoclonal.

Certain additional non-limiting embodiments of the present disclosureare directed to a method of preventing or reducing the occurrence ofrespiratory syncytial virus infection in a mammal by administering anyof the recombinant, live, attenuated viruses disclosed or otherwisecontemplated herein (or any of the pharmaceutical compositionscontaining same) to a mammal. In addition, any of the adjuvantsdescribed or otherwise contemplated herein may be administeredsimultaneously or partially or fully sequentially with the virus (orpharmaceutical composition containing same). In certain non-limitingembodiments, the mammal is susceptible to infection with RSV.

EXAMPLES

Examples are provided hereinbelow. However, the present disclosure is tobe understood to not be limited in its application to the specificexperimentation, results, and laboratory procedures disclosed herein.Rather, the Examples are simply provided as one of various embodimentsand are meant to be exemplary, not exhaustive.

Example 1

RSV is the most important viral respiratory pathogen of infancy andearly childhood, and yet there is no approved vaccine. One of the mainchallenges thus far has been to achieve strong efficacy and safetywithin one vaccine. A trial in the 1960s using formalin-inactivated RSVvaccine (FI-RSV) failed to protect against RSC and even resulted inenhanced disease severity upon exposure to wild type RSV (termedvaccine-enhanced disease or VED). In RSV-naïve children, VED is alsoinduced by many subunit approaches but not by live-attenuated vaccines.Live vaccines have additional advantages over non-replicating or subunitvaccines in that they induce immunologically-balanced, longer lastingprotection, including (but not limited to) mucosal immunity (naturalsite of infection) if administered intranasally.

To enable live vaccines with stable attenuation phenotypes, self-limited(single-round) live RSV viruses have been developed based on ablation ofessential genes (Matrix [M] or Fusion [F] protein) by providingfunctional replacements via a complementing production cell line. As ademonstration, by pseudotyping RSV with baculovirus entry/exit proteinGP64, it was possible to generate F-deleted yet infectious,single-round, RSV of high titer and with increased temperature stability(see, for example, US Patent Application Publication No. 2007/0104734and U.S. Pat. No. 7,588,770, incorporated supra). Importantly,production of these GP64 pseudotypes is completely independent of Ffunction. These viruses replicate their RNA genome at wildtype levels,generating abundant de novo viral antigens, but cannot spread beyond theinitial site of infection. A similar experimental vaccine based onablation of M induced robust immunity in an infant baboon model.

The spontaneous shift of the F protein from pre-fusion to post-fusionconformation during purification is believed to underlie the low levelsof neutralizing anti-F antibodies induced by vaccine preparations, andprobably also the loss of live RSV infectivity upon preparation andstorage. Recent publications have shown that the F protein can bereadily stabilized in the pre-fusion conformation through geneticchanges, and when used as a protein vaccine, induced a higher proportionof neutralizing anti-F antibodies in vivo (see, for example, Kwong etal., incorporated supra). However, F is essential to RSV, and thegenetically stabilized pre-fusion form (PreF) is no longer functional. Alive vaccine expressing PreF in place of native F (to drive the immuneresponse toward pre-fusion F without inducing VED in the RSV-naïvepopulation) is therefore not viable. Thus, to extend the advantageouspre-fusion F concept to the RSV-naïve population, an F-independentproduction system is needed that allows generation of live RSVexpressing PreF.

Thus, the present disclosure combines the pre-fusion F concept with theGP64 system previously developed by the inventor, as the GP64 system isF-independent and provides the tools to pursue these inventiveconcept(s).

Example 2

Oomens et al. (US Patent Application Publication No. 2007/0104734 andU.S. Pat. No. 7,588,770), incorporated supra) previously reported abaculovirus GP64 based complementation system that uniquely allowsgeneration of infectious F-deleted or F-compromised viruses from cDNA inGP64-expressing cells. These GP64-pseudotyped viruses could be amplifiedto high titer and were significantly more temperature stable thanwildtype RSV. Due to replacement of functional F with trans-complementedGP64, the viruses are infectious but self-limited and cannot spreadbeyond initially infected cells, thus constituting an attractivelive-attenuated platform.

The present disclosure exploits this F-independent, GP64 complementationsystem to generate a live RSV which solely expresses a pre-fusion Fprotein variant. Replacing the native, functional F gene with a geneencoding a pre-fusion stabilized F in a live virus provides a novelcombination of immunological benefits: it drives the anti-F responsetoward the pre-fusion F form, while also inducing a balanced responsethat includes cell-mediated immunity and avoidance of VED. In addition,a vaccine produced therefrom is single-round (self-limited) and thuscannot spread beyond the initially infected site, and is also moretemperature-stable. Absence of the CT from preF also provides anothersafety advantage: if the live vaccine virus were to attempt to mutatepreF in order to regain F function and virulence, absence of the CT willfurther prevent production of new progeny as the CT is required forvirus assembly. The resulting immunogenic composition/vaccine thus hasthe potential to exceed previous formulations in inducing a broadlyefficacious yet safe immune response for the RSV-naïve targetpopulation.

In certain non-limiting embodiments, the present disclosure uses theF-independent system based on a cell line that provides baculovirus GP64in trans to RSV, to generate live viruses that express a non-functionalpre-fusion F protein variant. In this manner, not only will the humoralarm be activated, leading to anti-pre-fusion F antibodies, but thecellular arm will also be activated, leading to anti-pre-fusion F CD8⁺lymphocytes, among others. Thus, contrary to protein-based vaccines, thelive immunogenic compositions/vaccines of the present disclosure willelicit both a humoral and a cellular response. Because the immunogeniccomposition/vaccine is based on RSV itself, immunity will also beinduced against all other RSV antigens, including (but not limited to)G. The F-independent system provides the baculovirus GP64 protein intrans, which results in a virus that is infectious but only for asingle-round, thereby making the vaccine incapable of inadvertentspreading throughout the lung of a recipient, and thus safer.

Three versions of pre-fusion stabilized F protein variants weregenerated for use in accordance with the present disclosure, as shown inFIG. 1. These pre-fusion stabilized F protein variants are based on thepreviously described preF fusion protein variant DS-Cav-1 (see, forexample, US 2015/0030622, US 2016/0031972, and US 2016/0046675,incorporated supra; and McLellan et al. (Science (2013) 342:592-598);the entire contents of which are expressly incorporated herein byreference). PreF^(ΔCT) is a membrane-anchored version that is expressedand anchored at the surface of infected cells; the amino acid sequenceof preF^(ΔCT) is represented by SEQ ID NO:2. PreF^(SEC) is a secretedversion that is secreted to the extracellular environment on infectedcells; the amino acid sequence of preF^(SEC) is represented by SEQ IDNO:3. PreF^(SEC/tag) is similar to PreF^(SEC) but contains an epitopetag for easy identification and detection; the amino acid sequence ofpreF^(SEC/tag) is represented by SEQ ID NO:4.

RSV viruses were then engineered with one of the three pre-fusionstabilized F variants of FIG. 1 inserted at either the 8^(th) or 6^(th)genome position, as shown in FIG. 2. Panel B of FIG. 2 depicts RSVgenomes with variants of pre-fusion stabilized F at the 8^(th) genomeposition, while Panel C of FIG. 2 depicts RSV genomes with variants ofpre-fusion stabilized F at the 6^(th) genome position. The 6th genomeposition was more highly expressed than the 8th genome position, toenhance the level of pre-fusion F. In addition, all of these virusesalso contained a GFP (Green Fluorescent Protein) marker gene fortracking and assay purposes. However, it will be understood that thepresence of GFP was simply for experimental purposes; GFP is notrequired to be present in the viruses of the present disclosure and canbe removed if necessary. All of these viruses also lacked expression ofthe secreted G protein (indicated as Gmem), which is a known virulencefactor. These vaccine viruses have been successfully generated, andstocks for each have also been generated with titers over 10⁷plaque-forming units per ml.

Successful expression of pre-fusion stabilized F protein variants fromthe engineered viruses generated as in FIG. 2 was verified, as shown inFIG. 3. Cells infected by virus RSV8-preF^(ΔCT) were incubated withanti-F and anti-G antibodies at 26 hours post-infection, ormock-infected as a negative control, and subjected to ELISA. Threeantibodies (provided by J S McLellan, The University of Texas at Austin)were used to detect the presence of F protein. The first antibody,Motavizumab (mota), detects both the pre-fusion and post-fusionconformation of F, while the second and third antibodies, D25 and 14402,are known to detect a different epitope specific only for the pre-fusionconformation of F (site Ø and site V). In addition, the G protein wasdetected at similar levels.

Therefore, as can be seen in FIGS. 4 and 5, abundant levels ofpre-fusion F were expressed at the surface of vaccine-virus infectedcells. In addition, Viruses RSV8-preF^(SEC) and RSV8-preF^(SEC/tag) havebeen similarly examined and were also demonstrated to express pre-fusionF.

In addition, viruses with pre-fusion F variants at position 6 willgenerate higher levels of pre-fusion F.

It was noted that pre-fusion F protein variants that contained thecytoplasmic tail of the F protein do not express at the cell surface aswell as the wildtype F protein. However, when the cytoplasmic tail wasremoved (such as (but not limited to) in virus RSV-preF^(ΔCT) describedin FIGS. 1-3), surface expression was improved to an expression levelsimilar to that observed for wildtype F protein. Therefore, while thescope of the present disclosure includes the use of F protein variantsboth with and without cytoplasmic tails, the absence of the cytoplasmictail can improve surface expression of the pre-fusion F protein variantand can thus assist with inducing immunity against the pre-fusion Fconformation.

Example 3

Removal of the cytoplasmic tail (CT) strongly improved cell-surfaceexpression of pre-fusion F. HEp2 cells were transfected with theindicated F expressing plasmids. The F open reading frames werecodon-optimized, as native F sequences express poorly in transfectedcells. To each well, a plasmid expressing NGFR-myc was added as atransfection control (NGFR-myc is expressed at the cell surface). At 46hours post-transfection, transfected cells were incubated with various Fantibodies or myc antibody as a control, and relative F surface levelswere detected using standard ELISA.

As shown in FIG. 3, full-length F and preF were detected at the cellsurface equally and at low levels. However, removal of the cytoplasmictail (CT) from preF (preF^(ΔCT)) led to a strong increase in surfaceexpression. As expected, surface expressed preF was recognized bypre-fusion-specific site Ø and V antibodies but not by a post-F specificantibody, demonstrating that preF^(ΔC) is in the pre-fusionconformation.

The vaccine candidate RSV6-preF^(ΔCT) induced high surface levels ofprefusion-F and G. HEp2 cells were infected with the indicated viruses.At 26 hours post-infection, infected cells were incubated with F, G, orN antibodies, and relative F and G surface levels were determined usingELISA (the N protein is an indicator of viral genomic replication and isshown for normalization purposes; to detect N, cells aredetergent-permeabilized).

As can be seen in FIG. 5, RSV6-preF^(ΔCT) induced higher surfaceexpression of prefusion F and G than a recombinant wild type (WT) virus.PreF is recognized by prefusion-specific site Ø and V antibodies but notby a post-F specific antibody, demonstrating that preF^(ΔCT) is in theprefusion conformation. As expected, RSV6-preF^(SEC) expresses G but notpreF at the plasma membrane.

In addition, vaccine candidate RSV6-preF^(SEC/tag) secreted high levelsof prefusion F. HEp2 cells were infected with the indicated viruses. At36 hours post-infection, supernatants of infected cells were harvestedand incubated on ELISA plates coated with the anti-tag antibodies for 1hour. Bound preF was then detected by ELISA using D25 and motavizumab asprimary antibodies.

As shown in FIG. 6, RSV6-preF^(SEC/tag) secreted high levels ofprefusion F, which was recognized by prefusion-specific antibody D25. Asexpected, RSV6-preF^(ΔCT) did not secrete any prefusion F protein intothe supernatant. Strong but slightly lower levels of prefusion F weredetected at 24 hours post-infection.

PreF expressing single cycle RSV was also shown to induce high levels ofanti-RSV antibodies in vivo. 96 well plates were coated with preF+G byinfecting HEp-2 cells with RSV6-preF^(ΔCT). At 26 hours post-infection,preF and G proteins were present at the cell surface in conformationallyaccurate (native) form. Pooled sera (n=3, collected at 3 weekspost-boost) from mice vaccinated prime/boost with RSV6-preF^(ΔCT) orRecWT virus were incubated on the coated ELISA plates, and antibodylevels were determined using ELISA.

As shown in FIG. 7, prime/boost vaccination with RSV6-preF^(ΔCT) inducedanti-RSV antibody levels similar to a wildtype virus, despite beinglimited to a single cycle of replication. (Note: The shown preF vaccinewas codon-optimized; codon-optimized and non-codon-optimized preFvaccines have been tested and gave similar results in mice). A low dose(0.5 million PFU/vaccination) of RSV6-preF^(ΔCT) induced equal levels ofantibodies as a high dose (1 million PFU/vaccination).

Example 4

In this Example, different preF-based single cycle RSV vaccines wereconstructed, and FIG. 8 graphically depicts a schematic overview of fiveexamples of different preF-based single cycle RSV vaccines constructedin accordance with the present disclosure. In addition toRSV6-preF^(ΔCT), two types of preF based vaccine candidates weregenerated. First, vaccines were generated in which known viral virulencefactors NS1 or NS2 have been moved to downstream positions todownregulate their expression levels. NS1 and NS2 are known to block thehost interferon response, and downregulating their expression isexpected to alter and improve the quality and longevity of the immuneresponse. As such, NS1 or NS2 were separately moved to the 8^(th) genomeposition. Second, vaccines were generated in which preF^(ΔCT) was movedto the 1st or 2nd genome position, for enhanced expression. The specificvaccine candidates generated are shown in FIG. 8 and include: Firstpanel, RSV6-preF^(ΔCT); second panel, RSV6-preF^(ΔCT)-NS1low; thirdpanel, RSV6-preF^(ΔCT)-NS2low; fourth panel, RSV1-preF^(ΔCT)-NS1low;fifth panel, RSV2-preF^(ΔCT)-NS2low.

RSV6-preF^(ΔCT) and RSV1-preF^(ΔCT)-NS1low have been examined in vitroand in vivo, as shown in FIGS. 9-12 and described in detail hereinbelow.

FIG. 9 graphically depicts that different preF RSV vaccines induced highlevels of preF and G protein at the cell surface. HEp-2 cells wereinfected with viruses RSV6-preF^(ΔCT) and RSV1-preF^(ΔCT)-NS1low. At 26hours post-infection, infected cells were incubated with anti-preF andanti-G antibodies, which were subsequently detected by standard ELISAmethod. Anti-N antibody was also used as an indicator for viral genomicreplication (N encapsulates the viral genome), and preF and G antibodylevels were determined without and with N level-based normalization.

Judged by N level, virus RSV1-preF^(ΔCT)-NS1low replicated to lowerlevels than RSV6-preF^(ΔCT). This matches the observation thatRSV1-preF^(ΔCT)-NS1low spreads a little more slowly through a cellculture, as seen by GFP expression. This is also consistent with theliterature and with NS1 having both anti-immune and pro-viral functions.Judged by normalized N levels, on a per virus basis, the two virusesgenerated very similar levels of preF and G proteins. This indicatesthat moving preF^(ΔCT) to the first genome position did not raise preFlevels, counter to expectations.

Next, the two viruses were examined in vivo. As can be seen in FIG. 10,different preF RSV vaccines induced high levels of preF-specific andG-specific antibodies. 96 well plates were coated with preF+G or preFalone as follows: HEP-2 cells were infected with RSV6-preF^(ΔCT) orRSV6-preF^(ΔCT)-AG (G gene removed). At 26 hours post-infection, eitherpreF+G or preF alone were present at the cell surface inconformationally accurate (native) form. Pooled sera (n=3, collected at3 weeks post-boost) from mice vaccinated prime/boost withRSV6-preF^(ΔCT) and RSV1-preF^(ΔCT)-NS1low were incubated on the coatedELISA plates (preF+G on the left; preF alone on the right), and antibodylevels were determined using standard ELISA method. Anti-G Ab L9 wasused to verify the absence of G protein in the preF-alone ELISA.

Prime/boost vaccination using two distinct single cycle preF vaccines(RSV6-preF^(ΔCT) and RSV1-preF^(ΔCT)-NS1low) induced both anti-G andanti-preF antibodies, despite being limited to a single cycle ofreplication. Whereas RSV1-preF^(ΔCT)-NS1low expressed in cell cultureoverall lower preF and G levels than RSV1-preF^(ΔCT), it induced equallevels of preF antibodies in vivo and moderately high levels of anti-Gantibodies than RSV1-preF^(ΔCT), indicative of potential vaccineadvantages. Lower NS1 levels may also increase immune memory.

The two distinct preF RSV vaccines were also shown to induce higherneutralizing antibody activity than a wildtype virus, despite beingsafe, single-cycle vaccines. Neutralizing anti-F antibodies from micevaccinated with RSV6-preF^(ΔCT), RSV1-preF^(ΔCT)-NS1low, or rec WT(ELISA) were tested. Three-fold dilutions of pooled mice sera (3 miceper pool; sera harvested 3 weeks post-boost) were incubated with 500 PFU(FIG. 11) or 250 PFU (FIG. 12) of virus RSV-AG-HRP, which lacks the Gprotein (allowing detection of F-specific neutralization) and containsthe HRP gene for detection. After a one hour incubation, virus-antibodysuspensions were incubated on HEp-2 cells for 1.5 hours. Inoculum wasremoved and cells incubated for a total of 48 hours post-infection(hpi). At 48 hpi, medium was replaced with standard ELISA substrate, andOD₄₅₀ was determined after 30 minutes as a measure of virus replication.

As shown in FIGS. 11 and 12, all viruses induced F-specificvirus-neutralizing antibodies. RSV6-preF^(ΔCT) andRSV1-preF^(ΔCT)-NS1low induced slightly higher levels of F-specificneutralization than rec WT virus, despite being limited to a singlecycle of virus replication. Neutralizing serum antibodies are a strongpredictor of in vivo protection potential. Because recWT virus waspreviously shown to protect mice from RSV challenge, the novel vaccinesof the present disclosure will protect animals in vivo and will also besafe, since they only replicate for a single round.

Thus, in accordance with the present disclosure, there have beenprovided compositions, as well as methods of producing and using same,which fully satisfy the objectives and advantages set forth hereinabove.Although the present disclosure has been described in conjunction withthe specific drawings, experimentation, results, and language set forthhereinabove, it is evident that many alternatives, modifications, andvariations will be apparent to those skilled in the art. Accordingly, itis intended to embrace all such alternatives, modifications, andvariations that fall within the spirit and broad scope of the presentdisclosure.

What is claimed is:
 1. A recombinant, live, attenuated virus of thePneumoviridae family comprising: a baculovirus GP64 envelopeglycoprotein or variant or fragment thereof, wherein the baculovirus G64envelope glycoprotein or variant or fragment thereof is capable ofmediating entry of the recombinant virus into a mammalian cell; and apolynucleotide encoding a respiratory syncytial virus (RSV) F proteinvariant or fragment thereof, wherein the RSV F protein variant orfragment thereof comprises at least one of the amino acid substitutionsS155C, S190F, V207L, and S290C when compared to the native RSV F proteinsequence of SEQ ID NO:1, wherein the at least one amino acidsubstitution stabilizes the RSV F protein variant or fragment thereof ina pre-fusion conformation.
 2. The recombinant, live, attenuated virus ofclaim 1, wherein the RSV F protein variant or fragment thereof comprisesthe amino acid substitutions S155C, S190F, V207L, and S290C whencompared to the native RSV F protein sequence of SEQ ID NO:1.
 3. Therecombinant, live, attenuated virus of claim 1, further defined as arecombinant respiratory syncytial virus.
 4. The recombinant, live,attenuated virus of claim 1, wherein the recombinant, live, attenuatedvirus maintains infective stability when stored at above 0° C. for atleast 3.5 days.
 5. The recombinant, live, attenuated virus of claim 1,further defined as an enveloped recombinant, live, attenuated virus. 6.The recombinant, live, attenuated virus of claim 1, wherein the virus iscapable of infecting a cell in a mammal but cannot transmit from saidcell to another cell in the mammal.
 7. The recombinant, live, attenuatedvirus of claim 1, wherein the polynucleotide encoding the RSV F proteinvariant or fragment thereof has been codon-optimized.
 8. Therecombinant, live, attenuated virus of claim 1, wherein thepolynucleotide encoding the RSV F protein variant or fragment thereofcomprises at least one of SEQ ID NOS:5-10.
 9. The recombinant, live,attenuated virus of claim 1, wherein the baculovirus GP64 envelopeglycoprotein or variant or fragment thereof is not encoded by the viralgenome but rather is obtained from a cell line from which the virus isproduced.
 10. The recombinant, live, attenuated virus of claim 1,wherein the baculovirus GP64 envelope glycoprotein or variant orfragment thereof is encoded by the viral genome.
 11. The recombinant,live, attenuated virus of claim 1, further encoding at least one of: anRSV NS1 protein or a variant or fragment thereof; an N protein or avariant or fragment thereof; a P protein or a variant or fragmentthereof; an M protein or a variant or fragment thereof; an SH protein ora variant or fragment thereof; a G protein or a variant or fragmentthereof; an M-2 protein or a variant or fragment thereof; an L proteinor a variant or fragment thereof; or any combination thereof.
 12. Therecombinant, live, attenuated virus of claim 1, further defined aslacking expression of at least one virulence factor encoded by the wildtype virus.
 13. The recombinant, live, attenuated virus of claim 1,wherein: (a) the baculovirus GP64 envelope glycoprotein or variant orfragment thereof comprises an ectodomain of the baculovirus GP64envelope glycoprotein; (b) the baculovirus GP64 envelope glycoprotein orvariant or fragment thereof comprises an ectodomain and a transmembranedomain of the baculovirus GP64 envelope glycoprotein; (c) thebaculovirus GP64 envelope glycoprotein or variant or fragment thereofcomprises a heterologous cytoplasmic tail; and/or (d) the baculovirusGP64 envelope glycoprotein or variant or fragment thereof comprises anamino acid sequence represented by SEQ ID NO:15.
 14. The recombinant,live, attenuated virus of claim 1, wherein: (a) the RSV F proteinvariant or fragment thereof is absent at least a portion of acytoplasmic tail of the native RSV F protein; (b) the RSV F proteinvariant or fragment thereof is absent at least a portion of atransmembrane domain and at least a portion of a cytoplasmic tail of thenative RSV F protein; (c) the RSV F protein variant or fragment thereoffurther comprises at least one of an epitope tag and a detectablemarker; and/or (d) the RSV F protein variant or fragment thereofcomprises an amino acid sequence represented by at least one of SEQ IDNOS:2-4.
 15. An isolated immunogenic composition, comprising: therecombinant, live, attenuated virus of claim
 1. 16. A pharmaceuticalcomposition, comprising: a therapeutically effective amount of arecombinant, live, attenuated virus of the Pneumoviridae family, thevirus comprising: a baculovirus GP64 envelope glycoprotein or variant orfragment thereof, wherein the baculovirus G64 envelope glycoprotein orvariant or fragment thereof is capable of mediating entry of therecombinant virus into a mammalian cell; and a polynucleotide encoding arespiratory syncytial virus (RSV) F protein variant or fragment thereof,wherein the RSV F protein variant or fragment thereof comprises at leastone of the amino acid substitutions S155C, S190F, V207L, and S290C whencompared to the native RSV F protein sequence of SEQ ID NO:1, whereinthe at least one amino acid substitution stabilizes the RSV F proteinvariant or fragment thereof in a pre-fusion conformation.
 17. A methodof producing the recombinant, live, attenuated virus of claim 1,comprising the steps of: culturing a cell line that expresses abaculovirus GP64 envelope glycoprotein or variant or fragment thereof,the cell line being transfected with at least one polynucleotideencoding RSV virus, wherein the RSV virus comprises an RSV F proteinvariant or fragment thereof that comprises at least one of the aminoacid substitutions S155C, 5190F, V207L, and S290C when compared to thenative RSV F protein sequence of SEQ ID NO:1, wherein the at least oneamino acid substitution stabilizes the RSV F protein variant or fragmentthereof in a pre-fusion conformation; and wherein the cell line iscultured under conditions that allow for production of the recombinant,live, attenuated virus.
 18. A method of eliciting an immune response ina mammal, comprising the step of: introducing into the mammal thepharmaceutical composition of claim 16.